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PubMed1980

Isolation and characterization of membranes from the cells of maize root tips.

A discontinuous sucrose density gradient was used to separate membrane fractions from a homogenate of maize root tips. Endoplasmic reticulum-, Golgi apparatus-, plasma membrane- and mitochondria-rich fractions were identified by their enzymic characteristics and by their appearance under the electron microscope. Maize roots were incubated in vivo with D-[U-14C]glucose, [Me-14C]choline chloride and diazotized [U-3H]sulphanilic acid. The pattern of incorporation of radioactivity into the various membrane fractions was investigated. Analyses of the polypeptide chains of the membrane fractions by SDS-polyacrylamide gel electrophoresis showed that the mitochondria-rich fraction had a different pattern of polypeptides from that of the other membrane fractions. The results are discussed in relation to the hypothesis of endomembrane flow and differentiation.

Amino Acids↗
PubMed1975

Production of carbon dioxide in vitro by blastocysts from intact and ovariectomized mice.

'Normal' and 'delayed implanting' mouse blastocysts were incubated in non-radioactive culture medium for various lengths of time (preincubation), and then placed in culture medium with (14-C)-glucose for 2 hr. The rate of embryonic CO-2 given off by the blastocysts. Normal embryos were unaffected by the length of preincubation. By contrast, 'delayed implantating' embryos had a low level of CO-2 production with short preincubations (i.e. less than 2 hr), but approached the normal range with longer preincubations (i.e. more than 8 hr) and thus, were 'activated ' in vitro. Incubation of the 'delayed implanting' embryos with actinomycin D prevented the expected increase in CO-2 production, indicating that synthesis of new RNA is necessary for their activation. Preincubation of normal and 'delayed implanting' blastocysts with oestradiol-17 beta and/ or progesterone had no effect on the level of CO-2 production, suggesting that the hormones do not directly stimulate or inhibit carbohydrate metabolism in the embryos.

Animals↗
PubMed1982

Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli.

The H+-ATPase (F1F0) of Escherichia coli was purified from cells labeled with either [35S]sulfate or [U-14C-D] glucose, and the molar ratio of subunits in the complex determined. The molar ratio was calculated from the radioactivity incorporated into each subunit, using either the subunit sulfur content or subunit molecular weight. These labeling experiments confirm an alpha 3 beta 3 gamma 1 delta 1 epsilon 1 ratio of subunits in F1, and indicate a chi 1 psi 2 omega 10 ratio of subunits in F0. The chi, psi, and omega designations used here refer to the subunits of F0 in order of decreasing molecular weight. Staining with Coomassie brilliant blue gave a reliable indication of the molar ratio of subunits in F1, but very erroneous values for each of the subunits of F0. We attempted to estimate the ratio of subunits in the native membrane, since the stoichiometry determined for the purified complex could be an anomaly of purification. These estimates were made after labeling cells with [35S]sulfate during amplification of the ATPase genes carried on a lambda transducing phage. The subunit ratios in the native membrane were reasonably close to those obtained with purified F1F0. We conclude that the stoichiometry determined reflects the composition of F1F0 in the native membrane. The most surprising conclusion from this study is that there are 10 +/- 1 omega ("proteolipid") subunits in each F1F0 complex. This is considerably more than had been assumed previously.

Adenosine Triphosphatases↗
PubMed1992

Effect of oxygen tension and lactate concentration on keratan sulphate and chondroitin sulphate biosynthesis in bovine cornea.

Calf cornea slices were incubated with [U-14C]glucose, in varying pO2 or lactate concentrations. Acid glycosaminoglycans were separated by ion-exchange chromatography after papain digestion. The percentage radioactivity incorporated into keratan sulphate increased markedly with decreased oxygen tension, whereas a concomitant relative decrease of the biosynthesis of glycosaminoglycuronans occurred. Similar results were obtained with increased lactate concentration. Our findings support the idea that keratan sulphate is a functional substitute for chondroitin sulphate in conditions of oxygen lack (Scott, J.E. and Haigh, M. (1988) J. Anat. 158, 95-108).

Animals↗
PubMed1999

[18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography localizes residual thyroid cancer in patients with negative diagnostic (131)I whole body scans and elevated serum thyroglobulin levels.

Progressive dedifferentiation of thyroid cancer cells leads to a loss of iodine-concentrating ability, with resultant false negative, whole body radioactive iodine scans in approximately 20% of all differentiated metastatic thyroid cancer lesions. We tested the hypothesis that all metastatic thyroid cancer lesions that did not concentrate iodine, but did produce thyroglobulin (Tg), could be localized by [18F]2-fluoro-2-deoxy-D-glucose (FDG)-positron emission tomography (PET). We performed FDG-PET on 37 patients with differentiated thyroid cancer after surgery and radioiodine ablation who had negative diagnostic 131I whole body scans during routine follow-up. Serum Tg, Tg autoantibodies, neck ultrasounds, and other clinically indicated imaging procedures were performed to detect residual disease. In those with elevated Tg levels, FDG-PET localized occult disease in 71%, was false positive in one, and was false negative in five patients. The majority of false negative FDG-PET occurred in patients with minimal cervical adenopathy. Surgical resections, biopsies, 131 therapy, and differentiation therapy were performed based on the PET results. The FDG-PET result changed the clinical management in 19 of the 37 patients. In patients with elevated Tg levels, FDG-PET had a positive predictive value of 92%. In patients with low Tg levels, FDG-PET had a negative predictive value of 93%. No FDG-PET scans were positive in stage I patients; however, they were always positive in stage IV patients with elevated Tg levels. An elevated TSH level (i.e. hypothyroidism) did not increase the ability to detect lesions. FDG-PET is able to localize residual thyroid cancer lesions in patients who have negative diagnostic 131I whole body scans and elevated Tg levels, although it was not sensitive enough to detect minimal residual disease in cervical nodes.

Adenocarcinoma, Follicular↗
PubMed1993

Effect of epinephrine on 86Rb efflux, 45Ca outflow and insulin release from pancreatic islets perifused in the presence of propranolol.

Pancreatic islets prelabelled with either 86Rb or 45Ca were perifused in the presence of propranolol (0.1 microM) and, when required, exposed to epinephrine (0.1 microM). In the absence of D-glucose, epinephrine failed to cause any obvious change in either 86Rb or 45Ca outflow. In the presence of 16.7 mM D-glucose, however, epinephrine lowered both 86Rb and 45Ca outflow, this coinciding with suppression of insulin release. Epinephrine also suppressed the increment in 86Rb outflow evoked by a rise in glucose-concentration from 8.3 to 16.7 mM. Epinephrine did not abolish the early fall in 45Ca efflux evoked by the administration of D-glucose (16.7 mM) to islets previously deprived of the hexose but, within the same experiments, impaired the secondary rise in effluent radioactivity. Likewise, epinephrine prevented the increase in 45Ca outflow provoked by a rise in hexose concentration from 8.3 to 16.7 mM. These findings are compatible with the recent proposal that epinephrine interferes with the entry of Ca2+ into the B-cell, as mediated by voltage-sensitive Ca2+ channels, but do not rule out a multifactorial coupling between the occupancy of alpha 2-adrenergic receptors and the eventual inhibition of insulin release.

Animals↗
PubMed1979

Variation in 125I-insulin absorption and blood glucose concentration.

The absorption of monocomponent porcine 125I-insulin Monotard and Isophane was studied in six insulin dependent diabetic patients over a period of 12 days. The absorption of insulin was measured as the disappearance of radioactivity from sites of injection. The daily 125I-insulin doses ranged from 20 to 48 IU between patients. The insulin absorbed varied considerably within and between patients. The range of individual daily absorbed insulin varied from 19 to 104 per cent of the 125I-insulin dose. A significant correlation (p less than 0.05) was found between insulin absorption and blood glucose concentration. Insulin absorption rates were relatively high before all hypoglycaemic episodes and reactive hyperglycaemia was only observed when relatively low insulin absorption rates followed the hypoglycaemic attack. The results show that lability in some insulin dependent diabetics is explained by variation in insulin absorption.

Absorption↗
PubMed1991

Potential errors due to aliasing in digital video analysis of quantitative autoradiography.

Digital image analysis of quantitative autoradiographic (QAR) films is widely used in neuroscience applications. Unless proper precautions are taken when autoradiographic images are converted to digital form they can be inadvertently modified by improper application of the sampling process. This type of modification is termed aliasing error and can cause nonexistent structures to appear in the reconstructed digital image, changing the apparent optical density values of the data. The theoretical basis of aliasing error is presented, along with examples of aliasing from optical resolution test patterns and 2-deoxy[14C]glucose (2-DG) experimental QAR images. We show that aliasing can change the apparent shape of structures, as well as the derived values obtained from QAR experiments. In an example with experimental 2-DG images, aliasing in the cerebellar cortex consistently underestimates tissue radioactivity levels in gray matter (P less than 0.001) and overestimates levels in adjacent white matter (P less than 0.001). Additional data transformations, such as the equations used for blood flow or glucose utilization, can, somewhat unpredictably, accentuate the errors introduced by aliasing. We present a discussion of the autoradiographic image features and electronic design that play a role in introducing aliasing errors and means by which aliasing can be recognized and minimized.

Animals↗
PubMed2001

18F-fluorodeoxyglucose accumulation in the heart, brain and skeletal muscle of rats; the influence of time after injection, depressed lipid metabolism and glucose-insulin.

BACKGROUND: To study the effect of lipid depressing drugs on (18)FDG myocardial concentration. The changes of (18)FDG uptake in myocardium, brain and skeletal muscle of rats were compared as influenced by acipimox, tyloxapol and glucose with insulin. MATERIAL AND METHODS: 5.55 MBq of (18)FDG were administered to Wistar rats. Control rats were killed 15, 30, 45 and 60 minutes following intravenous injection and the radioactivity concentration (cpm/g of tissue) in relation to injected cpm was determined in a well crystal adjusted to 511 KeV in order to check the time of maximal (18)FDG tissue uptake. The radioactivity in myocardium, skeletal muscle and brain in intact animals was compared with that of rats treated with tyloxapol (tritton WR 1339, 125 mg intravenously immediately before (18)FDG injection), acipimox (nicotinic acid derivative, 25 mg by stomach cannula 15 minutes before (18)FDG), or glucose with insulin (intravenous injection of 0.04 g and 0.04 UI immediately before (18)FDG). The animals were killed 45 minutes following (18)FDG injection. RESULTS: Tyloxapol and acipimox significantly elevated myocardial (18)FDG concentration (tyloxapol +37% and acipimox +48%), but the increase in (18)FDG concentration after glucose and insulin was slight and insignificant. The changes in skeletal muscle after lipid depressing agents were quite contrasting; the decrease in (18)FDG concentration was -74% after tyloxapol and -44% following acipimox administration. The accumulation of (18)FDG in brain was not influenced markedly by the drugs used or by glucose with insulin. CONCLUSION: The highest (18)FDG uptake in myocardium could be achieved by depressing the lipid metabolism and not by administration of glucose with insulin only. A marked increase in glucose accumulation in myocardium is not possible without previous shift from the utilisation of fatty acids. This finding is fully in agreement with present knowledge about energetic metabolism of myocardium.

Journal Article↗
PubMed1980

Glucosamine metabolism of herpes simplex virus infected cells. Inhibition of glycosylation by tunicamycin and 2-deoxy-D-glucose.

The formation of glucosamine-containing cell surface glycoproteins of herpes simplex virus (HSV) infected BMK cells was studied. Tunicamycin (TM) and 2-deoxy-D-glucose (DG) were used as inhibitors. With both inhibitors the multiplication of HSV was inhibited. DG markedly reduced cellular uptake of radioactively labelled glucosamine while TM interfered with the processing of glucosamine into TCA-insoluble material. Gel filtration chromatography on Sephadex G50 gel of cell surface material released by trypsin and further prepared by digestion with pronase indicated that TM and DG reduced the apparent high molecular weights of virus induced surface glycoproteins. In presence of DG the accumulation of a class of glucosamine-containing heterosaccharides (MW less than 3000) not present on DG-free HSV infected cells was observed. In TM treated cells virtually all surface heterosaccharides with molecular weights exceeding 3000 and containing glucosamine disappeared. Moreover, a component compatible with a lipid-linked oligosaccharide present in DG treated cells was not observed in HSV infected TM treated cells. The results exemplifies some different steps in glucosamine metabolism of virus induced cell surface glycoproteins differently affected by tunicamycin and 2-deoxy-D-glucose.

Animals↗
PubMed1979

Carbohydrate metabolism in C3H/HeJ (nonresponder) mice during endotoxic shock.

This study was undertaken to evaluate the hypoglycemic effects of endotoxin in C3H/HeJ (nonresponder) mice. Endotoxin from Salmonella enteritidis ser Typhimurium strain SR-11 was used and the median lethal dosed (LD50) for random-outbred Swiss-Webster mice and C3H/HeJ mice were 450 microgram and 3,000 microgram, respectively. At intervals after intraperitoneal injection of endotoxin (1 LD50) animals were killed, and blood glucose, liver glucose, and liver glycogen levels were killed, and blood glucose, liver glucose, and liver glycogen levels were measured. The time course of carbohydrate depletion in both strains of mice was almost identical. Little change from controls was noted, however, in nonresponder mice given the LD50 dose for normal responder mice. Passive transfer of plasma from C3H/HeJ mice appeared to protect conventional responder mice from the carbohydrate-depleting effects of endotoxin; whereas, passive transfer of peritoneal cells from C3HeB/FeJ responder mice to nonresponders appeared to sensitize C3H/HeJ mice to this effect. In order to evaluate clearance and detoxification of endotoxin in non-responder mice, 14 C-labeled lipopolysaccharide was prepared from bacteria grown in broth containing D-glucose-14 C(U). Mice were injected intravenously with labeled endotoxin, and blood, liver, spleen, kidney, heart, lung, and brain were counted for radioactivity at intervals after injection. Results from these tracer studies indicate that the clearance of lipopolysaccharide in nonresponder mice is slower than that seen in conventional animals. The results of this study further support the suggestion that endotoxin exerts its effects on carbohydrate metabolism via mediators resulting from endotoxin-cell surface interactions.

Animals↗
PubMed1984

Catecholamine physiology in the ovine fetus. III. Maternal and fetal response to acute maternal exercise.

A study was made of the effects of maternal exercise on fetal plasma concentrations of catecholamines in nine ewes with chronically catheterized singleton fetuses at 125 to 137 days' gestation. The ewes were subjected to acute treadmill exercise of 2.5 mph for 45 minutes with continuous recording of maternal and fetal blood pressures. Samples of arterial blood were obtained for measurement of catecholamines, glucose, and blood gases. Changes in blood flow in fetal organs in response to maternal exercise were assessed by injection of radioactive microspheres. The maternal plasma catecholamine responses were related to the severity of the exercise stress as indicated by the index of cardiac effort. The fetal responses did not correlate with maternal cardiac effort. A significant decrease in fetal Po2 with a moderate alkalosis occurred, accompanied by a significant elevation in circulating levels of norepinephrine. At the peak of exercise, there was an increase in fetal renal, adrenal, and placental blood flows, as compared to the control period.

Animals↗
PubMed1985

Metabolism of xylitol in dental plaque.

It has been reported previously that xylitol added to glucose used to challenge dental plaque in vivo caused a reduced acid formation. The aim of the present study was to approach the mechanism by which xylitol may affect glucose catabolism in plaque bacteria. Suspensions of freshly collected 4-day-old plaque bacteria were incubated, one batch with labeled xylitol, one with labeled glucose, in vitro at 37 degrees C. Samples of cells were taken out at time intervals, collected on paper discs and subjected to scintillation counting. It was observed that the plaque bacteria took up xylitol, the uptake increasing with incubation of more than 3-4 h, whereas the same cells took up glucose immediately. Cells which had taken up xylitol were extracted with boiling water, extracts concentrated and applied on thin-layer chromatography sheets. A radioactive component with mobility like xylitol-5-phosphate was isolated from the cell extracts, and also a component where labeled xylitol was associated with macromolecules. It is suggested that the accumulation of the metabolities within the cells inhibits glycolysis.

Carbon Radioisotopes↗
PubMed2000

Effects of nicotine and chlorisondamine on cerebral glucose utilization in immobilized and freely-moving rats.

Chlorisondamine blocks central nicotinic receptors for many weeks via an unknown mechanism. Intracerebroventricular administration of [(3)H]-chlorisondamine in rats results in an anatomically restricted and persistent intracellular accumulation of radioactivity. The initial aim of the present study was to test whether nicotinic receptor antagonism by chlorisondamine is also anatomically restricted. Male adult rats were pretreated several times with nicotine to avoid the disruptive effects of the drug seen in drug-naïve animals. They then received chlorisondamine (10 microg i. c.v.) or saline, and local cerebral glucose utilization (LCGU) was measured 4 weeks later after acute nicotine (0.4 mg kg(-1) s.c.) or saline administration. During testing, rats were partially immobilized. Nicotine significantly increased LCGU in the anteroventral thalamus and in superior colliculus. Chlorisondamine completely blocked the first of these effects. Chlorisondamine significantly reduced LCGU in the lateral habenula, substantia nigra pars compacta, ventral tegmental area, and cerebellar granular layer. The second experiment was of similar design, but the rats were not pre-exposed to nicotine, and were tested whilst freely-moving. Acute nicotine significantly increased LCGU in anteroventral thalamus, superior colliculus, medial habenula and dorsal lateral geniculate. Overall, however, nicotine significantly decreased LCGU. Most or all of the central effects of nicotine on LCGU were reversed by chlorisondamine given 4 weeks beforehand. These findings suggest that chlorisondamine blocks nicotinic effects widely within the brain. They also indicate that in freely-moving rats, nicotine can reduce or stimulate cerebral glucose utilization, depending on the brain area. British Journal of Pharmacology (2000) 129, 147 - 155

Animals↗
PubMed1987

Analysis of kinetic rate constants in [18F]fluorodeoxyglucose model using a least square fitting package SALS (statistical analysis with least squares).

The analysis of kinetic rate constants in the compartment model for [18F]fluorodeoxyglucose (FDG) was undertaken. Four kinetic rate constants were determined with a least square fitting package SALS (Statistical Analysis with Least Squares), using the measured data of 18F activity as a function of time. SALS calculations were found to be easy and quick with high precision. The rate constants and the curves fitted by the k3 and the k4 models were compared in situations with various degree of cerebral glucose metabolism during positron emission tomography (PET) studies. The k4 model in the determination of cerebral metabolic rate for glucose (CMRglu) was much superior than the k3 model in any given situation as it always underestimates the metabolic rate. However, the k3 model produces less variation when the cerebral radioactivity curve shows steady rising pattern.

Brain↗
PubMed1990

Non-enzymatic protein glycosylation: back-titration assay.

The glycosylation of albumin in vitro, as judged from the incorporation of D-[U-14C]glucose into trichloroacetic acid (TCA)-precipitable material represents a time-, temperature- and concentration-related process. It is markedly increased by NaCNBH3. A close correlation is observed between the radioactive data and the percentage of glycosylated albumin, as measured by affinity chromatography. The latter method, however, does not give information on the precise stoichiometry of the protein glycosylation. The relative extent of protein glycosylation can also be estimated by a back-titration procedure, the condensation of labelled D-glucose with the protein being inversely related to its prior degree of glycosylation. The back-titration assay was applied to plasma samples from normal and diabetic rats or human subjects and used to compare the glycosylation of albumin by distinct hexoses and hexose-phosphates. L-Glucose was found as efficient as D-glucose in causing albumin glycosylation and, hence, could conceivably be used to investigate in vivo changes in the intrinsic properties of extracellular proteins secondary to their glycosylation.

Animals↗
PubMed1963

PATTERNS OF OXIDATIVE ASSIMILATION IN STRAINS OF ACETOBACTER AND AZOTOBACTER.

Tomlinson, Geraldine A. (The University of British Columbia, Vancouver, B.C., Canada), and J. J. R. Campbell. Patterns of oxidative assimilation in strains of Acetobacter and Azotobacter. J. Bacteriol. 86:1165-1172. 1963.-Oxidative assimilation of glucose-U-C(14) was studied with washed-cell suspensions of Acetobacter aceti, A. xylinum, Azotobacter vinelandii, and A. agilis. The suggestion that oxidative assimilation is largely the incorporation of endogenously produced ammonia is tenable. A. aceti did not exhibit oxidative assimilation and it did not incorporate ammonia in the presence of glucose, alpha-ketoglutarate, or pyruvate. A. xylinum, A. vinelandii, and A. agilis incorporated C(14) into the nitrogenous fractions of the cell. The level of assimilation into A. xylinum was low due to the accumulation of extracellular cellulose, and the level of assimilation into the Azotobacter was low presumably because of the requirement of energy for nitrogen fixation. The Azotobacter were characterized by the presence of a high level of radioactivity in the cold trichloroacetic acid-soluble pool. None of the organisms accumulated compounds in the supernatant fluid that might be considered pacemakers in glucose oxidation, and this could be a contributing factor in the low level of assimilation.

Acetobacter↗